The invention concerns antibodies against the human La protein and then use for immunotargeting, in particular of tumor cells. The antibodies according to the invention are suited for use in the field of medicine, pharmacy and in biomedical research.
The human La protein (hLa) was originally disclosed as an auto-antigen in patients with systemic lupus erythematosus (S L E, Mattioli, M., Reichlin, M., 1974. Arthritis & Rheumatism; 17 (4): 421-29) and Sjögren syndrome (Alspaugh, M. A., Tan, E. M., 1975. J Clin Invest; 55 (5): 1067-73) and is known by the alternative name SS-B (Sjögren syndrome antigen B).
With 2·107 molecules per human cell it is an abundant protein which is found in all tissues. La has a functional role in RNA metabolism and as a RNA chaperon, it processes pre-tRNA precursor molecules and influences the exactness as well as the efficiency of the RNA polymerase III transcription in vitro (inter alia Gottlieb, E. et al., 1989 EMBO J; 8 (3): 841-50).
Chambers et al. (1988. J Biol. Chem., 263, 18043-18051) determined the amino acid sequence of La protein and 3 antigen epitope regions and made predictions about the regions which are involved in RNA binding.
The primary structure of hLa protein (FIG. 1A; SEQ ID No. 1) can be divided into three regions which form spatial domains that are independent of each other (see FIG. 1B). The La motif is N-terminal, followed by central RNA recognition motif (RNA recognition motif, RRM) which is also referred to as RRM1. These two domains form the N-terminal half of the protein and together referred to as LaN. The second half, LaC, contains the C-terminal RRM (RRM2) as well as an adjoining long, flexible element of about 80 AS that exhibits no secondary structure characteristics. According to Chambers et al. (1988), the N-terminal RRM1 is particularly immunogenic.
McNeilage et al. (1984. J. Clin. Rennet. Immunol. 15, 1-17., Clin. exp. Immunol., 1985, 62, 685-695) examine anti La antibodies formed by patients with autoimmune illnesses and which RNA and protein components are recognized by these antibodies. Scofield R H et al. examined the fine specificity of the autoimmune response against the Ro/SSA and La/SSB ribonucleoproteins (1999, Arthritis Rheum. 42 (2):199-209).
Chan E K. et al. (1987 J Exp Med 166 (6):1627-40) compare La epitopes which are recognized by human auto-antibodies and mouse antibodies against human and bovine La. For this purpose, 5 monoclonal mouse antibodies were produced which were obtained by immunization of mice with bovine La. A cross-reactivity of the mouse antibodies with murine La protein was not found.
Bachmann et al. (1990. Exp. Cell Res. 191, 171-180. 1991. Autoimmunity 9, 99-107. 1992 Autoimmunity 12, 37-45) describe that the auto-antigen La reaches the cell surface in UV-irradiated keranocytes and cells infected with herpes simplex type 1.
U.S. Pat. No. 5,457,029 describes a diagnostic test for detecting anti-La antibodies.
U.S. Pat. No. 4,751,181 discloses a procedure for producing the La protein antigen.
U.S. Pat. No. 4,784,942 discloses a murine anti-La antibody (hybridoma LA1, ATCC: HB-8609) which was obtained by immunization with bovine La protein.
Smith P R et al. (1985 J Immunol Methods 77(1):63-76) describe a murine anti-La antibody (SW5) which was obtained from rabbit by immunization with La protein.
Human monoclonal La antibodies were disclosed by Mamula M J (1989 J Immunol 143 (9):2923-8).
Offen D et al. (1990 J Autoimmune. 3 (6):701-13) describe murine anti-La antibodies directed against an ideotype (16/6 Id) spread in SLE patients.
In the following, the antibodies against the human La protein that are known in the art are summarized:                LalB5 Bachmann et al. PNAS USA vol. 83: 7770-7774, 1986;        8G3 and 9A5 Mamula et al. J Immunol 143 (9):2923-2928, 1989;        anti-human La Carmo-Fonseca et al. Exp Cell Res 185(1):73-85, 1989;        4B6 Tröster et al. J Autoimmunity 8 (6): 825-842, 1995;        SW1, SW3, SW5 Smith P R et al. J Immunol Methods. 77(1): 63-76, 1985;                    Pruijn et al. Eur J Biochem 232: 611-619, 1995;                        3B9 Tran et al. Arthritis Rheum 46(1): 202-208, 2002;        DAB4 Al Ejeh et al. Nucl Med Biol. 2009, 36(4): 395-402.        
All of these antibodies are monoclonal antibodies which bind to the human La protein. The CDR sequences of these antibodies are unknown except for SW5.
4B6 binds the La epitope with the sequence SKGRRFKGKGKGN (AS 330-343 of the human La protein, SEQ ID No. 2).
Al-Ejeh F. et al. (2007. Clin Cancer Res. 13 (18 Pt 2):5509s-5518s) describe that the La protein is expressed at increased levels in tumor cells. It was found that, with increasing DNA damage, the anti-La antibody 3B9 binds increasingly to tumor cells. It is described that the La antigen is cross-linked in dead malignant cells by transglutaminase 2. Based on this, Al-Ejeh F et al. (2007. Clin Cancer Res. 13 (18 Pt 2):5519s-5527s) describe in a mouse model the use of anti-La 3B9 for in vivo targeting of tumor cells. Targeting was further improved by concurrent cytostatic treatment. Al-Ejeh F et al. (2009. PLoS One. 4 (2):e4630) describe a radio immunotherapy in which tumor targeting is clone with the monoclonal anti-La antibody DAB4. The radio immunotherapy is carried out in combination with chemotherapy. WO 20081043148 A1 discloses also combination therapy of anti-La antibodies with cytostatic agents.
In Al-Ejeh F et al. (2009. PLoS One. 2009; 4 (2):e4558) the use of the anti-La antibody DAB4 is described for the detection of the tumor response to DNA-damaging chemotherapeutic agents.
A general problem of the therapeutic effectiveness of monoclonal antibodies in tumor treatment is the binding capacity of the antibodies to the cancer cells, i.e., the affinity of the antibodies and the selection of the suitable antigen which is bound by the antibodies. Specific tumor antigens mostly are not expressed in sufficient amounts on the cancer cells. In cancer cells that sufficiently express tumor antigens, the binding rate of the used antibodies is often not high enough. Moreover, with a molecular mass of about 150 kDa antibodies are limited in general with regard to tissue mobility. In this case, antibody fragments, like Fab, F(ab)2 or scFv (single chain variable fragments), on account of their clearly smaller size, have considerable advantages.
Bispecific antibodies, i.e., antibody derivatives of components of two different monoclonal antibodies, offer new possibilities for therapy concepts in cancer immunotherapy.
US 2005/0136050 A1 discloses bispecific antibodies which can bind to two different targets. They serve for recruiting human immune effector cells to a target antigen which is located on a target cell.
Quadromas are bispecific antibodies of the first generation and are comprised of a heavy chain and a light chain of two different monoclonal antibodies. The two arms of the antibody are each directed against different antigens. The Fc part is formed jointly of both heavy chains of the antibodies. With this construction it is, for example, possible to position the paratope of an antibody directed against a tumor antigen and the paratope of a further antibody directed against a lymphocyte antigen onto one arm of the bispecific antibody, respectively. It is so possible to form a three-cell complex resulting from the cells bound in each case by the different paratopes and the effector cell hound by the Fc part. In this context, an improved activation of the body's own immune cells arises generally relative to the tumor cells.
Bispecific antibodies of the newer generation are constructed of two different, scFv fragments. These are connected to each other by a linker peptide. Thus, a bispecific antibody can bind, for example, with one scFv to tumor cells and with the other scFv to effector cells.
When a paratope is directed against T cells, these cells can be also activated. With normal monoclonal antibodies this is not possible because T cells do not have Fc receptors. In addition, bispecific antibodies have a higher cytotoxic potential. They also bind to antigens which are expressed relatively weakly.
To this day, no bispecific antibodies have been approved for clinical use in humans.
Bispecific antibodies are known where an scFv binds to the CD3 complex on T cells, these are also called BiTE (bispecific T cell engager) (P. A. Baeuerle et. al., BiTE: Teaching antibodies to engage T cells for cancer therapy. Curr Opin Mol Ther 11, 2009, pages 22-30).
At the moment, two different BiTE antibodies are in clinical studies. Blinatumomab, an antibody directed against CD3 and CD19, is tested in patients in late phases of Non-Hodgkin lymphoma and in patients with acute lymphoblastic leukemia of the B cell line (B-ALL). MT110 is an antibody which is directed against CD3 and EpCAM (epithelial cell adhesion molecule) and is tested in patients with bronchial carcinoma and patients with gastrointestinal cancer diseases (R. Bargou en. al., Tumor regression in cancer patients by very low doses of a T cell-engaging antibody. Science 321, 2008, pages 974-977; K. Brischwein et al., MT110: a novel bispecific single chain antibody construct with high efficacy in eradicating established tumors. Mol Immunol 43, 2006, pages 1129-1143).
Not all monoclonal antibodies are suitable in the form of scFv fragments or for the construction of bispecific constructs. Particularly the affinity of the antibodies is decisive which is determined by the variable regions. Only particularly high-affinity antibodies are suited as scFv fragments because binding occurs at the respective antigen only with one pair of variable regions of the heavy and light chains, in contrast to the complete IgG antibodies which have two pairs of variable regions of the heavy and light chains.
For the treatment of carcinomas there is a need for new therapeutic concepts.
A big problem with immuno-targeting of cells, in particular in immunotherapy of tumors, is either the absence of specific targets or the loss of a specific target in some of the tumor cells. Therefore, no suitable targeting module could be developed up to now for many target cells.
The object of the invention is providing improved antibodies which bind universal target structures on the surface of tumor cells.